Lipid-binding Pocket Research Articles

The Molecular Basis of Vitamin E Retention: Structure of Human α-Tocopherol Transfer Protein

α-Tocopherol transfer protein (α-TTP) is a liver protein responsible for the selective retention of α-tocopherol from dietary vitamin E, which is a mixture of α, β, γ, and δ-tocopherols and the corresponding tocotrienols. The α-TTP-mediated transfer of α-tocopherol into nascent VLDL is the major determinant of plasma α-tocopherol levels in humans. Mutations in the α-TTP gene have been detected in patients suffering from low plasma α-tocopherol and ataxia with isolated vitamin E deficiency (AVED). The crystal structure of α-TTP reveals two conformations. In its closed tocopherol-charged form, a mobile helical surface segment seals the hydrophobic binding pocket. In the presence of detergents, an open conformation is observed, which probably represents the membrane-bound form. The selectivity of α-TTP for RRR-α-tocopherol is explained from the van der Waals contacts occurring in the lipid-binding pocket. Mapping the known mutations leading to AVED onto the crystal structure shows that no mutations occur directly in the binding pocket.

Phospholipid transfer is a prerequisite for PLTP-mediated HDL conversion.

Phospholipid transfer protein (PLTP) is an important regulator of high-density lipoprotein (HDL) metabolism. The two main functions of PLTP are transfer of phospholipids between lipoprotein particles and modulation of HDL size and composition in a process called HDL conversion. These PLTP-mediated processes are physiologically important in the transfer of surface remnants from lipolyzed triglyceride-rich lipoproteins to nascent HDL particles and in the generation of prebeta-HDL, the initial acceptor of excess peripheral cell cholesterol. The aim of the study presented here was to investigate the interrelationship between the two functions of PLTP. Plasma PLTP was chemically modified using diethylpyrocarbonate or ethylmercurithiosalicylate. The modified proteins displayed a dose-dependent decrease in phospholipid transfer activity and a parallel decrease in the ability to cause HDL conversion. Two recombinant PLTP mutant proteins, defective in phospholipid transfer activity due to a mutation in the N-terminal lipid-binding pocket, were produced, isolated, and incubated together with radioactively labeled HDL(3). HDL conversion was analyzed using three methods: native gradient gel electrophoresis, ultracentrifugation, and crossed immunoelectrophoresis. The results demonstrate that the mutant proteins (i) are able to induce only a modest increase in HDL particle size compared to the wild-type protein, (ii) are unable to release apoA-I from HDL(3), and (iii) do not generate prebeta-mobile particles following incubation with HDL(3). These data suggest that phospholipid transfer is a prerequisite for HDL conversion and demonstrate the close interrelationship between the two main activities of PLTP.

A lipid-binding domain in Wnt: a case of mistaken identity?

The alignments of sPLA2s and Wnts of Reichsman et al. [4xSequence homology between Wingless/Wnt-1 and a lipid-binding domain in secreted phospholipase A2. Reichsman, F, Moore, HM, and Cumberledge, S. Curr Biol. 1999; 9: R353–R355Abstract | Full Text | Full Text PDF | PubMedSee all References[4] and Barnes and Russell [17xA lipid-binding domain in Wnt: a case of mistaken identity?. Barnes, MR and Russell, RB. Curr Biol. 1999; 9: R717–R718Abstract | Full Text | Full Text PDF | PubMed | Scopus (1)See all References[17] were generated using two different paradigms. Barnes and Russell compared the physico-chemical properties of the amino acid residues; we used the Gonnet PAM 250 substitution matrix to evaluate amino acid similarities. PAM (percentage of acceptable point mutations) matrices have been widely recommended for detecting related proteins [[18]xSchwartz, RM and Dayhoff, MO. : 353–362See all References, [19]xThe significance of protein sequence similarities. Collins, JF, Coulson, AFW, and Lyall, A. Comp App Biosci. 1988; 4: 67–71PubMedSee all References, [20]xAmino acid substitution matrices from an information theoretic perspective. Altschul, S. J Mol Biol. 1991; 219: 555–565Crossref | PubMed | Scopus (389)See all References]. These substitution matrices are based on evolutionary relationships between proteins and are generated by analyzing point mutations in closely related proteins. Using the PAM matrices, we find that sPLA2 App-K49 residues 9–61 are more similar to the carboxy-terminal portion of Wingless than to the corresponding bovine sPLA2 sequences (44% versus 36% similarity). A total of 24 residues are conserved between sPLA2 and Wingless; 7 of these are cysteines. Note that App-K49 lacks the conserved Ala49, does not bind Ca2+ and has no catalytic activity, yet retains the ability to bind membranes and disrupt phospholipid vesicles [21xThe growing phospholipase A2 superfamily of signal transduction enzymes. Dennis, EA. Trends Biochem Sci. 1997; 22: 1–2Abstract | Full Text PDF | PubMed | Scopus (705)See all References[21].The first 60 residues of sPLA2 form the helix–loop–helix backbone that constitutes much of the lipid-binding pocket. The amino-terminal helix and loop are important for interfacial membrane binding, whereas residues in helix 2 contribute to the active site. As the rest of the App-K49 sequence has no similarity to Wingless, the two protein families probably have very different tertiary structures. Site-directed mutagenesis studies on sPLA2 [22xPhospholipase A2 engineering. The roles of disulfide bonds in structure, conformational stability, and catalytic function. Zho, H, Dupureur, CM, Zhang, X, and Tsai, MD. Biochemistry. 1995; 34: 15307–15314Crossref | PubMedSee all References[22] have shown that of the seven conserved disulphide bonds, only one, Cys29–Cys45, is required for lipase activity. Cys29 and Cys45 correspond to Cys428 and Cys445 in Wingless. These cysteines are conserved in the Wnts, and Cys445 is required for Wingless activity. Nonetheless, we agree that it is difficult to evaluate the significance of these sequence similarities without more information on Wnt function and structure.

N-terminal domain of apolipoprotein B has structural homology to lipovitellin and microsomal triglyceride transfer protein: a “lipid pocket” model for self-assembly of apoB-containing lipoprotein particles

The process of assembly of apolipoprotein (apo) B-containing lipoprotein particles occurs co-translationally after disulfide-dependent folding of the N-terminal domain of apoB but the mechanism is not understood. During a recent database search for protein sequences that contained similar amphipathic β strands to apoB-100, four vitellogenins, the precursor form of lipovitellin, an egg yolk lipoprotein, from chicken, frog, lamprey, and C. elegans appeared on the list of candidate proteins. The X-ray crystal structure of lamprey lipovitellin is known to contain a “lipid pocket” lined by antiparallel amphipathic β sheets. Here we report that the first 1000 residues of human apoB-100 (the α1 domain plus the first 200 residues of the β1 domain) have sequence and amphipathic motif homologies to the lipid-binding pocket of lamprey lipovitellin. We also show that most of the α1 domain of human apoB-100 has sequence and amphipathic motif homologies to human microsomal triglyceride transfer protein (MTP), a protein required for assembly of apoB-containing lipoproteins.▪Based upon these results, we suggest that an LV-like “proteolipid” intermediate containing a “lipid pocket” is formed by the N-terminal portion of apoB alone or, more likely, as a complex with MTP. This intermediate produces a lipid nidus required for assembly of apoB-containing lipoprotein particles; pocket expansion through the addition of amphipathic β strands from the β1 domain of apoB results in the formation of a progressively larger high density lipoprotein (HDL)-like, then very low density lipoprotein (VLDL)-like, spheroidal lipoprotein particle.—Segrest, J. P., M. K. Jones, and N. Dashti. N-terminal domain of apolipoprotein B has structural homology to lipovitellin and microsomal triglyceride transfer protein: a “lipid pocket” model for self-assembly of apoB-containing lipoprotein particles. J. Lipid Res. 1999. 40: 1401–1416.

Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K.

The catalytic contribution of His48 in the active site of porcine pancreatic phospholipase A2 was examined using site-directed mutagenesis. Replacement of His48 by lysine (H48K) gives rise to a protein having a distorted lipid binding pocket. Activity of this variant drops below the detection limit which is 10(7)-fold lower than that of the wild-type enzyme. On the other hand, the presence of glutamine (H48Q) or asparagine (H48N) at this position does not affect the structural integrity of the enzyme as can be derived from the preserved lipid binding properties of these variants. However, the substitutions H48Q and H48N strongly reduce the turnover number, i.e. by a factor of 10(5). Residual activity is totally lost after addition of a competitive inhibitor. We conclude that proper lipid binding on its own accelerates ester bond hydrolysis by a factor of 10(2). With the selected variants, we were also able to dissect the contribution of the hydrogen bond between Asp99 and His48 on conformational stability, being 5.2 kJ/mol. Another hydrogen bond with His48 is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol interacts with the enzyme. Its contribution to binding of the inhibitor in the presence of an interface was found to be 5.7 kJ/mol.

The three-dimensional structure of human bactericidal/permeability-increasing protein: Implications for understanding protein–lipopolysaccharide interactions

Gram-negative bacterial infections are often complicated by the inflammatory properties of lipopolysaccharides (LPS) on or released from the bacterial outer membrane. When present in the mammalian bloodstream, LPS can trigger a series of pathological changes, sometimes resulting in septic shock. Two related mammalian proteins, bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP), are known to affect the LPS-induced inflammatory response and are, therefore, of clinical interest. The recently determined three-dimensional structure of human BPI provides information on the overall protein fold, domain organization, and conserved regions of these two proteins. In addition, the discovery of two apolar lipid binding pockets in BPI indicates a possible site of interaction with LPS. The BPI structure is a powerful tool for the design of site-directed mutants, peptide mimetics/inhibitors, and BPI/LBP chimeras. These studies should help further define the functions of BPI and LBP, and their mechanism of interaction with LPS.

The implications of the structure of the bactericidal/permeability-increasing protein on the lipid-transfer function of the cholesteryl ester transfer protein

The cholesteryl ester transfer protein (CETP) is evolutionarily related to the bactericidal/permeability-increasing protein (BPI). The recently solved structure of BPI shows an elongated, boomerang-shaped molecule, with two hydrophobic pockets opening to its concave side. These pockets each contain a phospholipid molecule. A model of CETP, based on the recently solved crystal structure of BPI, provides the basis for interpreting functional studies on CETP. In this model, C-terminal residues 461–476, which were shown to be required for neutral lipid transfer between plasma lipoproteins, form an amphipathic helix covering the opening of the N-terminal pocket. A possible lipid-transfer mechanism for CETP, with the initial step involving the disordering of lipids in the lipoprotein surface, followed by the flipping and entry of a lipid molecule into the hydrophobic lipid-binding pocket, is hypothesized in light of structural evidence and recent studies.

The BPI/LBP family of proteins: a structural analysis of conserved regions.

Two related mammalian proteins, bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP), share high-affinity binding to lipopolysaccharide (LPS), a glycolipid found in the outer membrane of gram-negative bacteria. The recently determined crystal structure of human BPI permits a structure/function analysis, presented here, of the conserved regions of these two proteins sequences. In the seven known sequences of BPI and LBP, 102 residues are completely conserved and may be classified in terms of location, side-chain chemistry, and interactions with other residues. We find that the most highly conserved regions lie at the interfaces between the tertiary structural elements that help create two apolar lipid-binding pockets. Most of the conserved polar and charged residues appear to be involved in inter-residue interactions such as H-bonding. However, in both BPI and LBP a subset of conserved residues with positive charge (lysines 42, 48, 92, 95, and 99 of BPI) have no apparent structural role. These residues cluster at the tip of the NH2-terminal domain, and several coincide with residues known to affect LPS binding; thus, it seems likely that these residues make electrostatic interactions with negatively charged groups of LPS. Overall differences in charge and electrostatic potential between BPI and LBP suggest that BPI's bactericidal activity is related to the high positive charge of its NH2-terminal domain. A model of human LBP derived from the BPI structure provides a rational basis for future experiments, such as site-directed mutagenesis and inhibitor design.